ABSTRACT Dendritic cells (DCs) are key sentinel cells of the immune system that detect pathogens through pattern recognition receptors and orchestrate innate and adaptive immune responses. Conventional DCs (cDCs) efficiently present antigen to T lymphocytes, whereas plasmacytoid DCs (pDCs) specialize in virus-induced production of type I interferon. All DCs and their progenitors express the tyrosine kinase receptor Flt3, and its cytokine ligand Flt3L is necessary and sufficient for DC development. However, relatively little is known about the signaling pathways and molecules that regulate DC differentiation downstream of Flt3L. To dissect the molecular basis of DC differentiation, we implemented an unbiased forward genetics approach utilizing CRISPR/Cas9-based mutagenesis. In addition to known regulators, this approach yielded components of a protein complex that is enriched in DCs but has not been previously implicated in DC differentiation. The proposed project will explore the potential role of this complex in DC differentiation in vitro and in experimental animals (Aim 1) as well as the mechanism of its activity in DCs (Aim 2). If successful, these studies would provide novel insights into the molecular underpinnings of the DC differentiation, including lineage-specific signaling pathways that could be amenable to therapeutic targeting.